A sample detection device and method for nucleic acid qualitative analysis based on PCR

Abstract

The present invention provides a sample detection device and method for nucleic acid qualitative analysis based on PCR, wherein the detection device includes a light source, a signal acquisition module, a data processing module, and a display module. The light source is configured to continuously illuminate the sample area through the optical path module, and the signal acquisition module is configured to continuously collect signals and convert electrical signals within a detection time threshold after the sample area is illuminated. The data processing module is configured to respond to the electrical signal to form a fluorescence increase index, which is the ratio of electrical signals at different time points, set a threshold for the fluorescence increase index, and compare the obtained fluorescence increase index with the threshold. When the fluorescence increase index has an increasing trend and is greater than the first threshold, the detection result is obtained in advance.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic medical devices, and in particular to a sample detection device and method for nucleic acid qualitative analysis based on PCR. Background Art

[0002] In the prior art, nucleic acid sample detection based on polymerase chain reaction (PCR) generally involves centralized collection of samples, which are then sent to the laboratory for centralized testing. The main reason is that the reaction takes a long time, the steps are complicated, and professional laboratory personnel are required to complete the detection. However, this detection method takes a long time to detect, is extremely expensive, and is also prone to clustered infections. Therefore, many companies have launched antigen detection kits based on immunochromatography methods, allowing residents to self-test at home to determine the infection status, which can solve the huge cost and clustered infection problems. However, there are still limitations when using antigen detection kits based on immunochromatography methods, because they can only perform antigen-antibody pairing tests on virus samples that are already present in the sample. Generally, when the virus content in the sample is very small, it will be judged as negative, which has no advantage for early diagnosis of patients. Detection of nucleic acid samples based on polymerase chain reaction is still the safest detection method, so how to perform nucleic acid sample detection on the polymerase chain reaction platform and solve the above problems is a problem we have to face.

[0003] In the prior art, some products have initially solved the above problems. However, since the method of fluorescence detection is based on polymerase chain reaction, the amplification cycle is generally determined for the same test item when the fluorescence detection is used for nucleic acid sample detection. When the amplification time reaches the amplification cycle, the fluorescence detection is performed again, and the fluorescence detection result is used as the basis for judgment. Figure 3 As shown in the figure, it is a trend chart of the change of the electrical signal value during the positive sample amplification process. The detected electrical signal will continue to accumulate with the change of time. However, due to the light decay phenomenon, the displayed electrical signal result will weaken before reaching the predetermined detection time threshold. The final electrical signal data used as the basis for judging the result is different from the actual result, which can easily lead to the misjudgment of negative results. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a sample detection device and method for nucleic acid qualitative analysis based on PCR:

[0005] The detection device includes a sample tube and a detector, wherein the sample tube includes a blank area and a sample area, and before the detector starts detection, the sample to be detected enters the sample area through the blank area. The detector includes a housing, a sample holder, an optical path module, and a hardware module. The hardware module includes a light source, a signal acquisition module, a data processing module, and a display module.

[0006] The light source is configured to continuously illuminate the sample area through the optical path module, and the signal acquisition module is configured to continuously detect the sample area within the detection time threshold H and at intervals at the detection time point T after the sample area is illuminated. n Receive signal S from the optical path module n The data processing module is configured to respond to the signal S n The fluorescence increase index K is formed, and the fluorescence increase index K is the signal S at different time points n The fluorescence increase index is compared with the first threshold value. If K shows an increasing trend and is greater than the first threshold value, the detection is stopped and a first result is generated and indicated through the display module.

[0007] In the first case, the fluorescence increase index K = S n / S1, compare the fluorescence increase index with the first threshold and the second threshold, and set the second threshold to be less than the first threshold. If K is increasing and greater than the first threshold, stop the test and generate and indicate the first result; otherwise, repeat the comparison until T n Equal to H, if K is between the first threshold and the second threshold, stop detecting, generate and indicate the second result; if K is not between the first threshold and the second threshold, stop detecting, generate and indicate the third result.

[0008] Furthermore, the comparison is repeated until T n is equal to H, if K remains unchanged and S n If it is greater than a third threshold, detection is stopped and a fourth result is generated and indicated, wherein the third threshold is an electrical signal threshold.

[0009] In the second case, the fluorescence increase index K = S n / S n-1 , compare the fluorescence increase index with the first threshold, compare the signal S n and the second threshold, where the second threshold is the electrical signal threshold. If K is increasing and is greater than the first threshold, the detection is stopped and the first result is generated and indicated; otherwise, the comparison is repeated until T n is equal to H, if K is less than the first threshold and S n If the value is less than the second threshold, the detection is stopped and the second result is generated and indicated; otherwise, the detection is stopped and the third result is generated and indicated.

[0010] Furthermore, the comparison is repeated until T n is equal to H, if K is less than the first threshold and S n If it is greater than a second threshold, detection is stopped and a fourth result is generated and indicated, wherein the second threshold is an electrical signal threshold.

[0011] Furthermore, K being an increasing trend means that, in the second case, after the fluorescence increase index K is greater than the first threshold, the fluorescence increase index K at adjacent detection time points is greater than the first threshold.

[0012] Specifically, the detection device also includes an indicator light, the first result is displayed as a positive indicator light, the second result is displayed as a negative indicator light, the third result is displayed as an invalid indicator light, and the fourth result is displayed as a warning indicator light.

[0013] Furthermore, the sample support also includes a temperature control component. The sample support is accommodated in the L-shaped notch of the optical path module, and the temperature control component is arranged on the opposite side of the vertical surface of the L-shaped notch.

[0014] Furthermore, the signal acquisition module and the data processing module are respectively arranged on different printed circuit boards and are detachably connected via a first connecting component and a second connecting component.

[0015] The sample detection method for nucleic acid qualitative analysis based on PCR requires the execution of nucleic acid detection to induce a constant temperature amplification reaction, which causes a signal to continuously appear over time. n Before reaching the detection time threshold H, determine the signal S n The fluorescence increase index K is defined as the ratio of the signal Sn at different time points. The determined fluorescence increase index is compared with the upper threshold value. When it is determined that the fluorescence increase index has only an increasing trend and is greater than the upper threshold value, it indicates a detection result.

[0016] Furthermore, before confirming that the fluorescence increase index has only an increasing trend and is greater than the upper threshold, the comparison is repeated. n When the detection time threshold H is reached, it is determined that the fluorescence increase index K has at most an increasing trend, but is less than the upper threshold, indicating a detection result.

[0017] Furthermore, when the detection time point T n When the detection time threshold H is reached, it is determined that the fluorescence increase index does not have any trend, indicating a detection result.

[0018] The sample detection device for nucleic acid qualitative analysis based on PCR and the detection device used in the sample detection method for nucleic acid qualitative analysis based on PCR also include a fast charging deception protocol chip and a power supply interface. The power supply voltage is obtained through the fast charging deception protocol chip and the power supply interface, and the input voltage is determined to be greater than the fourth threshold through the hardware module, and an instruction is given to start detection, otherwise no detection is performed.

[0019] The beneficial effects of the present invention include: 1. For the sample detection device based on PCR for nucleic acid qualitative analysis, by designing the detection and calculation logic, a positive / affirmative test result can be obtained before the detection time reaches the detection time threshold, thereby avoiding the test result being affected by the light decay phenomenon. 2. For the same test item, different test conclusions can be drawn for different types of samples to be tested, providing users with more effective test conclusions to help users determine the next steps. 3. By setting a fast charging decoy protocol chip, a more stable voltage / current is provided for nucleic acid detection, ensuring that the amplification reaction and fluorescent signal detection have a stable detection environment. 4. Provide a small nucleic acid detection device that can obtain test results earlier, realize personal and family testing, and solve the problem of clustered infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 One of the schematic diagrams of the detection device of the present invention.

[0021] Figure 2 One of the schematic diagrams of the sample support of the detection device of the present invention.

[0022] Figure 3 Signal change trend diagram during the amplification process of positive samples (with light decay phenomenon).

[0023] Figure 4 Fluorescence increase index trend diagram of influenza A virus non-positive / negative samples of the present invention.

[0024] Figure 5 The fluorescence increase index trend diagram of influenza A virus positive samples of the present invention.

[0025] Figure 6 Fluorescence increase index trend diagram of influenza A virus non-positive / non-negative / amplified samples of the present invention.

[0026] Figure 7 Fluorescence increase index trend diagram of influenza A virus non-positive / non-negative / invalid samples of the present invention.

[0027] Figure 8 Fluorescence increase index trend diagram of influenza B virus non-positive / negative samples of the present invention.

[0028] Fig. 9 The fluorescence increase index trend diagram of influenza B virus positive samples of the present invention.

[0029] Fig.10 Fluorescence increase index trend diagram of influenza B virus non-positive / non-negative / amplified samples of the present invention.

[0030] Fig.11 Fluorescence increase index trend diagram of influenza B virus non-positive / non-negative / invalid samples of the present invention. Implementation

[0032] Example 1

[0033] like Figure 1 As shown, the sample detection device for nucleic acid qualitative analysis based on PCR includes a sample tube 1 and a detector 2, the sample tube 1 includes a blank area 11 and a sample area 12, and the detector 2 includes a housing 21, a sample holder 22, an optical path module 23, and a hardware module 24. During the specific detection, the sample to be detected is added to the sample tube 1, the sample to be detected enters the sample area 12 through the blank area 11, the sample tube is placed in the sample holder of the detector, the housing of the detector is closed, and the detection begins.

[0034] The detector includes a housing, a sample holder, an optical path module and a hardware module. The length of the detector is within 10 cm, the width is within 6 cm, and the height is within 10 cm, but preferably the length is within 9 cm, the width is within 5.5 cm, and the height is within 9 cm. The sample holder has at most 3 positions for placing sample tubes. Of course, there can be more positions, but the size of the instrument needs to be larger. The optical path module 23 has an L-shaped notch, and the sample holder is just accommodated in the L-shaped notch 231. When the sample tube is placed in the sample holder, the lower half of the sample tube is accommodated in the sample holder. The sample holder has a light-transmitting hole corresponding to the vertical surface of the L-shaped notch. The light-transmitting hole allows the excitation light to enter the sample holder so that the lower half of the sample tube can be illuminated by the light source.

[0035] The hardware module 24 includes a light source (not shown), a signal acquisition module 241, a data processing module 242 and a display module 243. The light source is configured to continuously illuminate the sample area 12 through the optical path module 23; the signal acquisition module is configured to collect the fluorescence signal emitted from the sample area 12 after the sample area 12 is illuminated, convert it into an electrical signal and output it to the data processing module; the data processing module 242 is configured to calculate and compare the received electrical signal and generate a detection result; the display module 24 is configured to receive the detection result generated by the data processing module and display it. The light source is located at the bottom of the optical path module 23. When the light source emits light, the excitation light illuminates the sample area 12 of the sample tube 1 through the reflective structure of the optical path module 23. The fluorescence in the sample to be tested in the sample area 12 is excited to generate and emit fluorescence. The emitted fluorescence is collected by the acquisition module 241. The path of the fluorescence being collected is perpendicular to the path of the excitation light illuminating the sample area. From the illumination of the light source to the collection of the fluorescence, the effective light forms an inverted U-shaped light path in the optical path module. Because the optical path is in an inverted U shape and the optical path is folded multiple times, the detection device as a whole has an ultra-small structural size. After the signal acquisition module collects the fluorescent signal, it converts it into an electrical signal, which is output to the data processing module for calculation and comparison to generate the detection result. The display module indicates or displays the detection result through an indicator light or a display screen.

[0036] The detection time threshold of the detection item is set to H, that is, the maximum time for amplification and detection of a single nucleic acid sample. The threshold is set differently according to different detection items. After the light source illuminates the sample area through the optical path module, the inverted U-shaped optical path continues to exist, and the acquisition module 241 continuously collects signals at intervals within the detection time threshold, that is, at the first time point T1, the signal S1 is detected, at the second time point T2, the signal S2 is detected, at the third time point T3, the signal S3 is detected, and at the nth time point T n Detection signal S n The interval between each time point is set differently according to different detection items, for example, it is generally 20 seconds.

[0037] The fluorescence increase index of the signal collected by the signal acquisition module is set to K. The threshold of K is set according to different projects. For the same project, the threshold of the fluorescence increase index K is fixed. The fluorescence increase index K refers to the electrical signal S detected at different time points. n Ratio, for example, K = S n / S1 or K=S n / S n-1 The data processing module is configured to respond to the electrical signal S received from the signal acquisition module. n The fluorescence increase index K is formed, and the corresponding calculation and comparison are performed to generate the result. For example, when the signal S1 at the time point T1 is received, K1=S1 / S1 is calculated, and after the detection signal S2 at the time point T2 is received, K2=S2 / S1 is calculated, and when the signal S3 at the time point T3 is received, K3=S3 / S1 is calculated, and it is compared whether K3>K2>K1, but it is not required that all fluorescence increase indexes are greater than the previous number. In other words, the fluorescence increase index has an increasing trend, and it is compared whether K3 is greater than the set threshold; or in the above process, K1=S2 / S1, K2=S3 / S2, and it is compared whether K2>K1, but it is not required that all fluorescence increase indexes are greater than the previous number. In other words, the fluorescence increase index has an increasing trend, and it is compared whether K2 is greater than the set threshold. If the above process obtains a positive result, the data processing module outputs a signal to stop the detection and generates a positive result, which is indicated by the display module to be obtained by the user. If the above process obtains a negative result, the data processing module does not output a signal to stop detection, the signal acquisition module continues to collect and convert signals at intervals, and the data processing module continues to calculate and compare the received signals.

[0038] For example, after the above process, when the signal S4 at time point T4 is received, K4=S4 / S1 or K3=S4 / S3 is calculated. Compare whether K1 to K4 are increasing trends and whether K4 is greater than the set threshold; or compare whether K1 to K3 are increasing trends and whether K3 is greater than the set threshold. If so, the data processing module outputs a signal to stop detection and generates a positive result, which is indicated by the display module for the user to obtain. If not, the signal acquisition module continues to collect and convert signals at intervals, and the data processing module continues to calculate and compare the received signals.

[0039] On the other hand, the fluorescence increase index K has a first threshold and a second threshold, and the second threshold is less than the first threshold. When K1=S1 / S1, K2=S2 / S1, K3=S3 / S1, K4=S4 / S1 and no positive result is generated, continue to collect signals, and when the signal S5 at the T5 time point is received, calculate K5=S5 / S1. Compare whether T5 is equal to the threshold H of the overall detection time, and whether K1, K2, K3, and K4 are less than the first threshold but greater than the second threshold, and K1 to K2 have at most an increasing trend. If so, a signal to stop detection is output and a negative result is generated, which is indicated by the display module to be obtained by the user. If T5 is not yet equal to H, the signal acquisition module continues to collect and convert signals at intervals, and the data processing module continues to calculate and compare the received signals. If the above result has not appeared when T5 is equal to H, a signal to stop detection is output and an invalid result is generated, which is indicated by the display module to be obtained by the user. In other cases, when T5 is equal to H, a comparison is made to determine whether K1, K2, K3, and K4 remain roughly unchanged, that is, the data are approximately equal, and the signal S5 is greater than the set electrical signal threshold, then a signal to stop the detection is output and a warning result is generated, which is indicated by the display module to be obtained by the user, informing the user that the sample is a non-instant sample and should be re-collected for detection. The electrical signal threshold varies depending on the project. For a specific project, the electrical signal threshold means that when the electrical signal value exceeds a certain value, the sample detected is unlikely to be a negative sample.

[0040] When K1=S2 / S1, K2=S3 / S2, K3=S4 / S3 and no positive result is generated, continue to collect signals, and after receiving the signal S5 at the time point T5, calculate K4=S5 / S4. Compare whether T5 is equal to the threshold value H of the overall detection time, and whether K1, K2, K3, and K4 have no regular change trend, and K1 to K4 are all less than the first threshold value. If so, output a signal to stop detection and generate a negative result, which is indicated by the display module for the user to obtain. If T5 is not yet equal to the threshold value H of the overall detection time, the data processing module does not output a signal to stop detection, the acquisition module continues to collect and convert signals at intervals, and the data processing module continues to calculate and compare the received signals. If T5 is already equal to H, and the above result does not appear, output a signal to stop detection and generate an invalid result, which is indicated by the display module for the user to obtain. In other cases, when T5 is equal to H, the added determination signal S5 is determined to be greater than the electrical signal threshold. If so, a signal to stop detection is output and a warning result is generated. The result is indicated by the display module so that the user can obtain it, informing the user that the sample is a non-immediate sample and should be re-collected for detection.

[0041] In other cases where K1=S2 / S1, K2=S3 / S2, K3=S4 / S3 produce positive results, continue to collect signals, and after receiving the signal S5 at time point T5, calculate K4=S5 / S4 and compare whether K4 is greater than the first threshold. If so, the data processing module outputs a signal to stop detection and produces a positive result, which is indicated by the display module for the user to obtain. If not, the signal acquisition module continues to collect and convert signals at intervals, and the data processing module continues to calculate and compare the received signals.

[0042] The detector 2 also includes an indicator light. The positive result is displayed as a positive indicator light, the negative result is displayed as a negative indicator light, the invalid result is displayed as an invalid indicator light, and the warning result is displayed as a warning indicator light. Figure 2 As shown, the sample holder 22 of the detector 2 also includes a temperature control component 220. The detector controls the ambient temperature of the sample holder through the temperature control component, rapidly heats up at the beginning of the detection, and monitors the temperature change. The temperature can be controlled within a certain range at different detection time points, and the ambient temperature control is stopped at the end of the detection. The temperature control component is arranged on the opposite side of the vertical surface of the L-shaped notch 231, that is, the surface on the sample holder that does not have a light-transmitting hole but is parallel to the vertical surface of the L-shaped notch. In order to prevent the detection process from being affected by temperature, the signal acquisition module and the data processing module are respectively arranged on different printed circuit boards in conjunction with the temperature control component, and are detachably and electrically fixedly connected through a first connecting component and a second connecting component. The first connecting component and the second connecting component can be a pin header and a female header, and of course, can also be other devices that can achieve the same effect.

[0043] Example 2

[0044] The following uses the influenza A virus detection kit as an example to illustrate the detection method based on PCR for isothermal amplification of nucleic acids and detection of the results.

[0045] Step 1: Add the sample to be tested into the sample area of ​​the sample tube through the blank area of ​​the sample tube. The sample to be tested is an uncertain sample, which can be a positive sample with influenza A virus or a non-positive sample without influenza A virus. Cover the lid of the sample tube, place the sample tube in the sample holder of the detector, and close the lid of the detector housing.

[0046] Step 2: Operate the detector through a mobile phone APP or directly on the detector to start the detection work. The sample to be tested is fully mixed with the preset reagent in the sample area to re-dissolve the preset reagent. The preset reagent contains a fluorescent probe. The constant temperature amplification reaction begins, and the light source illuminates the sample area through the optical path module.

[0047] Step 3: After the detection work starts, the signal acquisition module collects the fluorescence signal at a time interval of 20 seconds and converts it into an electrical signal and outputs it to the data processing module.

[0048] Step 4: The data processing module obtains the first electrical signal result 464 20 seconds after the start of the detection, and calculates K1=464 / 464=1.00, compares K1 with the threshold value 2.50, and concludes that K1 is less than the threshold value.

[0049] Step 5: Repeat steps 3 and 4. The data processing module obtains the second electrical signal result 468 after 40 seconds, and calculates the second fluorescence increase index K2=468 / 464=1.01. By comparing K2 with the threshold value 2.50 and K2 with K1, it is concluded that K2 is less than the threshold value but greater than K1.

[0050] Step 6: Repeat step 5 until 17 minutes and 40 seconds later, the data processing module obtains the electrical signal result 1168 and calculates the fifty-third fluorescence increase index K 53 =1168 / 464=2.52, compare K 53 with a threshold of 2.50, and K1 to K 53 The changing trend of K 53 Greater than the threshold and K1 to K 53 The conclusion is an increasing trend.

[0051] Step 7: The data processing module outputs the test result that the sample to be tested is positive, and the detector stops the detection work. Table 1 shows the value of the electrical signal and the value of the fluorescence increase index K obtained by the data processing module in this process. Figure 5The figure shows the trend of the fluorescence increase index in the process. It can be seen that although the fluorescence increase index is not increasing all the time, it has an increasing trend.

[0052] Table 1 Influenza A virus positive sample test results of the present invention

[0053]

[0054] The above is one of the situations that may occur during the test process, that is, it will only occur when the test result is positive. When the test result is not positive, the first to third steps are the same, and the fourth to seventh steps are as follows.

[0055] Step 4: The data processing module obtains the first electrical signal result 398 20 seconds after the start of the detection, and calculates K1=398 / 398=1.00, compares K1 with the threshold value 2.50, and concludes that K1 is less than the threshold value.

[0056] Step 5: Repeat steps 3 and 4. The data processing module obtains the second electrical signal result 399 after 40 seconds, and calculates the second fluorescence increase index K2=399 / 398=1.00. By comparing K2 with the threshold value 2.50 and K2 with K1, it is concluded that K2 is less than the threshold value and K has not changed.

[0057] Step 6: Repeat step 5 until the accumulated detection time reaches the time threshold of 40 minutes. The data processing module obtains the electrical signal result 608 and calculates the 120th fluorescence increase index K. 120 =608 / 398=1.53, compare K 120 with a threshold of 2.50, and K1 to K 120 The changing trend of K1 to K 120 The conclusion is that they are all less than the threshold value of 2.50 and K has an increasing trend.

[0058] Step 7: The data processing module outputs a non-positive test result for the sample to be tested, and the detector stops testing. Table 2 shows the value of the electrical signal and the value of the fluorescence increase index K obtained by the data processing module during this process. Figure 4 The figure shows the trend of the fluorescence increase index in the process. It can be seen that although the fluorescence increase index is not increasing all the time, it has an increasing trend.

[0059] Table 2 Influenza A virus non-positive / negative sample detection results of the present invention

[0060]

[0061] The above is the second situation that may occur during the test process, that is, it only occurs when the test result is non-positive. When the test result is negative, the first to third steps are the same, and the fourth to seventh steps are as follows.

[0062] Step 4: The data processing module obtains the first electrical signal result 398 20 seconds after the start of the test, and calculates K1=398 / 398=1.00, compares K1 with the small threshold 1.00 and the large threshold 2.50, and concludes that K1 is equal to the small threshold and is less than the large threshold.

[0063] Step 5: Repeat steps 3 and 4. The data processing module obtains the second electrical signal result 399 after 40 seconds, and calculates the second fluorescence increase index K2=399 / 398=1.00. By comparing K2 with the small threshold value 1.00 and the large threshold value 2.50, as well as the change trend from K1 to K2, it is concluded that K2 is between the small threshold value and the large threshold value and there is no change trend from K1 to K2.

[0064] Step 6: Repeat step 5 until the accumulated detection time reaches the time threshold of 40 minutes. The data processing module obtains the electrical signal result 608 and calculates the 120th fluorescence increase index K. 120 =608 / 398=1.53, compare K 120 with a small threshold of 1.00 and a large threshold of 2.50, and analyzes K1 to K 120 The trend of K1 to K 120 are between the small threshold and the large threshold, and K1 to K 120 There is only one conclusion about the signal accumulation trend, and signal accumulation means that the signal gradually strengthens.

[0065] Step 7: The data processing module outputs the test result that the sample to be tested is negative, and the detector stops the test. Table 2 shows the value of the electrical signal and the value of the fluorescence increase index K obtained by the data processing module in this process. Figure 4 The figure shows the trend of the fluorescence increase index in the process. It can be seen that although the fluorescence increase index is not increasing all the time, it has an increasing trend.

[0066] The above is the third situation that may occur during the test process, that is, it will only occur when the test result is negative. When the test result is non-negative or non-positive, the first to third steps are the same, and the fourth to seventh steps are as follows.

[0067] Step 4: The data processing module obtains the first electrical signal result 1691 20 seconds after the start of the test, and calculates K1=1691 / 1691=1.00, compares K1 with the small threshold 1.00, and concludes that K1 is equal to the small threshold. Or compares K1 with the small threshold 1.00 and the large threshold 2.50, and concludes that K1 is equal to the small threshold and is less than the large threshold. Or compares K1 with the small threshold 1.00, and compares the electrical signal with the electrical signal threshold 1000, and concludes that K1 is equal to the small threshold and the electrical signal is greater than the electrical signal threshold.

[0068] Step 5: Repeat Step 3 and Step 4. The data processing module obtains the second electrical signal result 1698 after 40 seconds, and calculates the second fluorescence increase index K2=1698 / 1691=1.00. Compare K2 with the small threshold value 1.00, and K2 with K1, and conclude that K2 is approximately equal to the small threshold value and approximately equal to K1. Alternatively, compare K2 with the small threshold value 1.00 and the large threshold value 2.50, and K2 with K1, and conclude that K2 is approximately equal to the small threshold value and approximately equal to K1. Alternatively, compare K2 with the small threshold value 1.00, and compare the electrical signal with the electrical signal threshold value 1000, and conclude that K2 is approximately equal to the small threshold value and the electrical signal is greater than the electrical signal threshold value.

[0069] Step 6: Repeat step 5 until the accumulated detection time reaches the time threshold of 40 minutes. The data processing module obtains the electrical signal result 1671 and calculates the 120th fluorescence increase index K. 120 =1671 / 1691=0.99, compare K 120 With the small threshold of 1.00, we finally get K1 to K 120 The conclusion is that they are approximately equal to the small threshold value of 1.00. Or compare K 120 with a small threshold of 1.00 and a large threshold of 2.50, and analyzes K1 to K 120 The trend of K1 to K 120 are approximately equal to the small threshold 1.00, and K1 to K 120 Conclusion of no signal accumulation trend. Or compare K 120 The value of K is obtained by comparing the value of the electric signal with the small threshold value 1.00 and the value of the electric signal with the electric signal threshold value 1000. 120 The conclusion that the electrical signal is approximately equal to the small threshold and is greater than the electrical signal threshold.

[0070] Step 7: The data processing module outputs a non-positive test result for the sample to be tested, and the detector stops testing. Or the data processing module outputs a non-positive and non-negative test result for the sample to be tested, and the detector stops testing. Or the data processing module outputs a test result that the sample to be tested is an amplified sample, and the detector stops testing. Table 3 shows the electrical signal values ​​and the fluorescence increase index K values ​​obtained by the data processing module during this process. Figure 6 The figure shows the trend of the fluorescence increase index during the process. It can be seen that the fluorescence increase index does not show an increasing trend, but tends to remain unchanged.

[0071] Table 3 Influenza A virus non-positive / non-negative / amplified sample detection results of the present invention

[0072]

[0073] The above is the fourth situation that may occur during the test process, that is, it will only appear if the test result is non-positive, or non-negative and non-positive. In this case, other results will appear, which is the same as the first to third steps of the above process, and the subsequent description is as follows. This is the fifth situation.

[0074] Step 4: The data processing module obtains the first electrical signal result 137 20 seconds after the start of the test, and calculates K1=137 / 137=1.00, compares K2 with the small threshold 1.00, and concludes that K1 is equal to the small threshold. Or compares K1 with the small threshold 1.00 and the large threshold 2.50, and concludes that K1 is equal to the small threshold and is less than the large threshold. Or compares K1 with the small threshold 1.00, and compares the electrical signal with the electrical signal threshold 1000, and concludes that K1 is equal to the small threshold and the electrical signal is less than the electrical signal threshold.

[0075] Step 5: Repeat Step 3 and Step 4. The data processing module obtains the second electrical signal result 208 after 40 seconds, and calculates the second fluorescence increase index K2=208 / 137=1.52. Compare K2 with the small threshold value 1.00, and K2 with K1, and conclude that K2 is greater than the small threshold value and approximately greater than K1. Alternatively, compare K2 with the small threshold value 1.00 and the large threshold value 2.50, and K2 with K1, and conclude that K2 is greater than the small threshold value and less than the large threshold value, and K2 is greater than K1. Alternatively, compare K2 with the small threshold value 1.00, and compare the electrical signal with the electrical signal threshold value 1000, and conclude that K2 is greater than the small threshold value and the electrical signal is less than the electrical signal threshold value.

[0076] Step 6: Repeat step 5 until the accumulated detection time reaches the time threshold of 40 minutes. The data processing module obtains the electrical signal result 152 and calculates the 120th fluorescence increase index K.120 =152 / 137=1.11, compare K 120 with a small threshold of 1.00, and analyzing K1 to K 120 The trend of K1 to K 120 The conclusion that K is approximately greater than or equal to the small threshold of 1.00 and K does not have an obvious change pattern. Or compare K 120 with a small threshold of 1.00 and a large threshold of 2.50, and analyzes K1 to K 120 The trend of K1 to K 120 are approximately greater than or equal to the small threshold of 1.00 and less than the large threshold of 2.50, and K1 to K 120 There is no obvious conclusion of change pattern. Or compare K 120 With a small threshold of 1.00, analyze K1 to K 120 By comparing the electrical signal with the electrical signal threshold of 1000, we can get K 120 is greater than the small threshold and the electrical signal is less than the electrical signal threshold and K1 to K 120 There is no conclusion with obvious rules of change.

[0077] Step 7: The data processing module outputs a non-positive test result for the sample to be tested, and the detector stops testing. Or the data processing module outputs a non-positive and non-negative test result for the sample to be tested, and the detector stops testing. Or the data processing module outputs a test result for the sample to be tested as an invalid sample, and the detector stops testing. Table 4 shows the electrical signal values ​​and the fluorescence increase index K values ​​obtained by the data processing module during this process. Figure 7 The figure shows the trend of the fluorescence increase index in the process. It can be seen that the fluorescence increase index does not have an obvious change pattern.

[0078] Table 4 Influenza A virus non-positive / non-negative / invalid sample test results of the present invention

[0079]

[0080] Example 3

[0081] The following uses the influenza B virus detection kit as an example to illustrate the detection method based on PCR for isothermal amplification of nucleic acids and detection of the results.

[0082] Step 1: Add the sample to be tested into the sample area of ​​the sample tube through the blank area of ​​the sample tube. The sample to be tested is an uncertain sample, which can be a positive sample with influenza B virus or a non-positive sample without influenza B virus. Cover the lid of the sample tube, place the sample tube in the sample holder of the detector, and close the lid of the detector housing.

[0083] Step 2: Operate the detector through a mobile phone APP or directly on the detector to start the detection work. The sample to be tested is fully mixed with the preset reagent in the sample area to re-dissolve the preset reagent. The preset reagent contains a fluorescent probe. The constant temperature amplification reaction begins, and the light source illuminates the sample area through the optical path module.

[0084] Step 3: After the detection work starts, the signal acquisition module collects the fluorescence signal at a time interval of 20 seconds and converts it into an electrical signal and outputs it to the data processing module.

[0085] Step 4: The data processing module obtains the first electrical signal result 542 after 20 seconds from the start of detection, and obtains the second electrical signal result 545 after 40 seconds, calculates the fluorescence increase index K1=545 / 542=1.01, compares K1 with the threshold value 1.05, and concludes that K1 is less than the threshold value.

[0086] Step 5: The data processing module obtains the third electrical signal result 545 60 seconds after the start of the detection, calculates the fluorescence increase index K2=545 / 545=1.00, compares K1 with the threshold value 1.05, and concludes that K2 is less than the threshold value.

[0087] Step 6: The data processing module obtains the time-varying electrical signal at 20-second intervals, calculates the corresponding fluorescence increase index K, compares K with the threshold, and analyzes the overall change trend of K.

[0088] Step 7: Repeat step 6 until 15 minutes and 20 seconds later, the data processing module obtains the electrical signal result 674 and calculates the 45th fluorescence increase index K 45 =674 / 635=1.06, compare K 45 with a threshold of 1.05, and K1 to K 45 The overall trend of K 45 Greater than the threshold and K1 to K 45 The conclusion is that the overall trend is increasing.

[0089] Step 8: The data processing module outputs the test result that the sample to be tested is positive, and the detector stops the detection work. Table 5 shows the value of the electrical signal and the value of the fluorescence increase index K obtained by the data processing module in this process. Fig. 9 The figure shows the trend of the fluorescence increase index in the process. It can be seen that although the fluorescence increase index is not increasing all the time, it has an obvious increasing trend.

[0090] Table 5 Influenza B virus positive sample test results of the present invention

[0091]

[0092] The above is one of the situations that may occur during the detection process, that is, it will only occur when the test result is positive. When the test result is not positive, it is the same as the first to fourth steps of the above process, and the follow-up is described as follows.

[0093] Step 7: Repeat step 6 until the accumulated detection time reaches the time threshold of 35 minutes. The data processing module obtains the electrical signal result 600 and calculates the corresponding fluorescence increase index K. 104 =600 / 603=1.00, compare K 104 with a threshold of 1.05, and K1 to K 104 The overall trend of K 104 Less than the threshold and K1 to K 104 There is no conclusion that there is a regular trend of change overall. Or compare S1 to S 105 The value of S1 to S 105 The conclusion is that the value is less than the electrical signal threshold of 800.

[0094] Step 8: The data processing module outputs a non-positive test result for the sample to be tested, and the detector stops testing. Alternatively, the data processing module outputs a negative test result for the sample to be tested, and the detector stops testing. Table 6 shows the electrical signal values ​​and the fluorescence increase index K values ​​obtained by the data processing module during this process. Figure 8 The figure shows the trend of the fluorescence increase index in the process. It can be seen that the fluorescence increase index has no obvious change pattern, that is, it does not have an increasing trend.

[0095] Table 6 Influenza B virus non-positive / negative sample detection results of the present invention

[0096]

[0097] The above is the second situation that may occur during the detection process, which is the detection process of non-positive test results. In other cases, there are different results, which are the same as the first to third steps of the above process, and the follow-up is described as follows.

[0098] Step 4: The data processing module obtains the first electrical signal result 2024 after 20 seconds from the start of detection, and obtains the second electrical signal result 2023 after 40 seconds, calculates the fluorescence increase index K1=2023 / 2024=1.00, compares K1 with the threshold value 1.05, and concludes that K1 is less than the threshold value.

[0099] Step 5: The data processing module obtains the third electrical signal result 2054 60 seconds after the start of the detection, calculates the fluorescence increase index K2=2054 / 2023=1.02, compares K1 with the threshold value 1.05, and concludes that K2 is less than the threshold value.

[0100] Step 6: The data processing module obtains the time-varying electrical signal at 20-second intervals, calculates the corresponding fluorescence increase index K, compares K with the threshold, and analyzes the overall change trend of K.

[0101] Step 7: Repeat step 6 until the accumulated detection time reaches the time threshold of 35 minutes. The data processing module obtains the electrical signal result 2115 and calculates the corresponding fluorescence increase index K. 104 =2115 / 2118=1.00, compare K 104 with a threshold of 1.05, and K1 to K 104 The overall trend of K 104 Less than the threshold and K1 to K 104 There is no overall regular conclusion. Or compare S1 to S 105 The value of S1 to S 105 The conclusion is that the value is greater than the electrical signal threshold of 800.

[0102] Step 8: The data processing module outputs a non-positive test result for the sample to be tested, and the detector stops testing. Or the data processing module outputs a test result for the sample to be tested that has been amplified, and the detector stops testing. Table 7 shows the value of the electrical signal and the value of the fluorescence increase index K obtained by the data processing module during this process. Fig.10 The figure shows the trend of the fluorescence increase index in the process. It can be seen that the fluorescence increase index has no obvious change pattern, that is, it does not have an increasing trend.

[0103] Table 7 Influenza B virus non-positive / non-negative / amplified sample detection results of the present invention

[0104]

[0105] The above is the third situation that may occur during the detection process. If none of the above three situations occur, the data processing module outputs a non-positive test result for the sample to be tested, and the detector stops the detection work. Alternatively, the data processing module outputs a test result that the sample to be tested is an invalid sample, and the detector stops the detection work. Table 8 shows the value of the electrical signal and the value of the fluorescence increase index K obtained by the data processing module during this process. Fig.11 The figure shows the trend of the fluorescence increase index in the process. It can be seen that the fluorescence increase index has no obvious change pattern, that is, it does not have an increasing trend.

[0106] Table 8 Influenza B virus non-positive / non-negative / invalid sample test results of the present invention

[0107]

[0108] The above is the fourth situation that may occur during the detection process. Of course, there are other situations, that is, in another situation of the first situation, which is the fifth situation, the following steps are added between the seventh step and the eighth step:

[0109] Repeat step 7. After 15 minutes and 40 seconds, the data processing module obtains the electrical signal result 715 and calculates the 46th fluorescence increase index K. 46 =715 / 674=1.06, compare K 46 With the threshold value of 1.05, we get K 46 Conclusion greater than the threshold.

[0110] Example 4

[0111] According to the detection principle of the above-mentioned embodiment 2 and embodiment 3, the nucleic acid sample is qualitatively detected based on PCR. There is no special limitation on the monitoring device. However, the detection step requires that after the nucleic acid sample is collected, the fluorescence signal during the amplification is detected while the isothermal amplification reaction is performed:

[0112] For positive nucleic acid samples, the isothermal amplification reaction causes the fluorescence signal to accumulate in a time-varying manner, that is, within a certain period of time, the longer the isothermal amplification reaction time, the stronger the intensity of the detected fluorescence signal. n Before reaching the set detection time threshold H (for example, 30 minutes), determine the detected electrical signal S n The fluorescence increase index K is defined as the signal S at different time points. n Ratio, for example, K = S n / S1 or K=S n / S n-1 The determined fluorescence increase index is compared with the set upper threshold value (i.e., the fluorescence increase index value that a non-positive sample will not reach). When it is determined that the obtained fluorescence increase index has only an increasing trend and the most recent fluorescence increase index is greater than the upper threshold value, it indicates a positive test result.

[0113] For non-positive nucleic acid samples, before it is determined that the fluorescence increase index has only one increasing trend and is greater than the upper threshold, the determined fluorescence increase index is repeatedly compared with the upper threshold, and the change trend of the fluorescence increase index is analyzed. The same sample as in Example 2, Case 3, is used for detection, and the obtained electrical signal trend graph is the same as Figure 4 Similarly, when the detection time point reaches the detection time threshold, it is determined that the fluorescence increase index has only one signal increasing trend, and the fluorescence increase index is less than the upper threshold, indicating that the test result is non-positive or negative. The same sample as in the second case of Example 3 is used for detection, and the obtained electrical signal trend diagram is the same as Figure 8 Similarly, when the detection time point reaches the detection time threshold, it is determined that the fluorescence increase index has no regular change trend, that is, there is no signal increasing trend, and the fluorescence increase index is less than the upper limit threshold, indicating that the test result is non-positive; moreover, it is determined that all electrical signals are less than the electrical signal threshold, indicating that the test result is negative.

[0114] The same samples as those in cases 3 to 5 of Example 2 are used for testing, and the results similar to those in the corresponding step 6 are obtained; or the same samples as those in cases 2 to 4 of Example 3 are used for testing, and the results similar to those in the corresponding step 7 are obtained. That is, when the detection time point reaches the time threshold, it has been determined that the fluorescence increase index does not show at most one increasing trend, which means that the detection result is not a non-positive result.

[0115] Example 5

[0116] In order to ensure that the amplification reaction and the fluorescence signal detection can have the most stable detection environment, the detection devices of the above-mentioned embodiments 1 to 4 are powered by an external fast charging power adapter, and a fast charging deception protocol chip is configured in the detection device. The detection device obtains the fast charging power (12V / 1A) on the external power adapter through the fast charging deception protocol. The principle is the same as the fast charging deception of the current mobile phone. After the power interface of the detection device obtains the voltage of the external adapter, the hardware module of the detection device divides the obtained voltage and monitors it. The calculation formula is as follows:

[0117]

[0118] Among them, V refers to the calculated voltage divider value, ADC refers to the obtained adapter voltage value, and R1 and R2 are the resistance values ​​of the voltage divider resistors. If the calculated voltage divider value V is less than the set voltage threshold, which refers to the minimum voltage divider value that allows the detection to have a stable detection environment, such as 2V, the detection device indicates that no detection will be performed, which means that the power adapter is not suitable and needs to be replaced with a suitable power adapter. For example, this can be prompted by a flashing indicator light. If the calculated voltage V is greater than the set voltage threshold, such as 2V, the detection device indicates to start detection, which means that the power adapter is suitable. For example, this can be prompted by a constant light indicator light.

[0119] The above specific implementation modes are used to explain the present invention rather than to limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A sample detection device for nucleic acid qualitative analysis based on PCR, comprising a sample tube and a detector, characterized in that: The sample tube includes a blank area and a sample area, the sample enters the sample area via the blank area, and the detector includes a housing, a sample support, an optical path module, and a hardware module; The hardware module includes a light source, a signal acquisition module, a data processing module and a display module; The light source is configured to continuously illuminate the sample area through the optical path module, and the signal acquisition module is configured to continuously detect the sample area within a detection time threshold H and at intervals at detection time points T after the sample area is illuminated. n Receive the signal S from the optical path module n ; The data processing module is configured to respond to the signal S n The fluorescence increase index K is formed, and the fluorescence increase index K is the signal S at different time points. n The ratio of the fluorescence increase index to the first threshold is: If K shows an increasing trend and is greater than the first threshold, the detection is stopped, and a first result is generated and indicated through the display module.

2. A sample detection device for nucleic acid qualitative analysis based on PCR according to claim 1, characterized in that: The fluorescence increase index K = S n / S1, comparing the fluorescence increase index with a first threshold and a second threshold, the second threshold being less than the first threshold: If K is in an increasing trend and is greater than a first threshold, the detection is stopped, and a first result is generated and indicated; Otherwise, the comparison is repeated until T n Equal to H, if K is between the first threshold and the second threshold, stop detecting, generate and indicate the second result; if K is not between the first threshold and the second threshold, stop detecting, generate and indicate the third result.

3. A sample detection device for nucleic acid qualitative analysis based on PCR according to claim 2, characterized in that: Repeat the comparison until T n is equal to H, if K remains unchanged and S n If the value is greater than the third threshold, detection is stopped and a fourth result is generated and indicated.

4. A sample detection device for nucleic acid qualitative analysis based on PCR according to claim 1, characterized in that: The fluorescence increase index K = S n / S n-1 , compare the fluorescence increase index with the first threshold, signal S n With the second threshold: If K is in an increasing trend and is greater than a first threshold, the detection is stopped, and a first result is generated and indicated; Otherwise, the comparison is repeated until T n is equal to H, if K is less than the first threshold and S n If the value is less than a second threshold, the detection is stopped, and a second result is generated and indicated; Otherwise, the detection is stopped and a third result is generated and indicated.

5. A sample detection device for nucleic acid qualitative analysis based on PCR according to claim 4, characterized in that: Repeat the comparison until T n is equal to H, if K is less than the first threshold and S n If the value is greater than the second threshold, detection is stopped and a fourth result is generated and indicated.

6. A sample detection device for nucleic acid qualitative analysis based on PCR according to claim 4, characterized in that: The increasing trend of K means that after the fluorescence increase index K is greater than the first threshold, the fluorescence increase index K at adjacent detection time points is greater than the first threshold.

7. A sample detection device for nucleic acid qualitative analysis based on PCR according to claim 1, characterized in that: The detection device also includes an indicator light, the first result is displayed as a positive indicator light, and the second result is displayed as a negative indicator light.

8. A sample detection device for nucleic acid qualitative analysis based on PCR according to claim 2 or 4, characterized in that: The detection device also includes an indicator light, the first result is displayed as a positive indicator light, the second result is displayed as a negative indicator light, and the third result is displayed as an invalid indicator light.

9. A sample detection device for nucleic acid qualitative analysis based on PCR according to claim 3 or 5, characterized in that: The detection device also includes an indicator light, and the fourth result is displayed as a warning indicator light.

10. A sample detection device for nucleic acid qualitative analysis based on PCR according to any one of claims 1 to 6, characterized in that: The sample support further comprises a temperature control component. The sample support is accommodated in the L-shaped notch of the optical path module. The temperature control component is arranged on the opposite side of the vertical surface of the L-shaped notch.

11. A sample detection device for nucleic acid qualitative analysis based on PCR according to claim 10, characterized in that: The signal acquisition module and the data processing module are respectively arranged on different printed circuit boards and are detachably connected via a first connecting component and a second connecting component.

12. A sample detection method for nucleic acid qualitative analysis based on PCR, wherein the detection is performed to cause a constant temperature amplification reaction, wherein the constant temperature amplification reaction causes a signal to continuously appear over time, characterized in that: At the detection time point T n Before reaching the detection time threshold H, determine the signal S n The fluorescence increase index K is defined as the signal S at different time points. n The ratio of the determined fluorescence increase index and the upper limit threshold is compared; when it is determined that the fluorescence increase index has only an increasing trend and is greater than the upper limit threshold, it indicates the detection result.

13. The sample detection method for nucleic acid qualitative analysis based on PCR according to claim 12, characterized in that: Before it is determined that the fluorescence increase index has only an increasing trend and is greater than the upper threshold, the comparison is repeated; when the detection time point T n When the detection time threshold H is reached, it is determined that the fluorescence increase index has at most an increasing trend, but is less than an upper threshold, indicating the detection result.

14. The sample detection method for nucleic acid qualitative analysis based on PCR according to claim 12, characterized in that: When the detection time point T n When the detection time threshold H is reached, it is determined that the fluorescence increase index does not have any trend, indicating the detection result.

15. A sample detection device for nucleic acid qualitative analysis based on PCR according to any one of claims 1 to 6, characterized in that: The detection device includes a fast charging deception protocol chip and a power supply interface. The power supply voltage is obtained through the fast charging deception protocol chip and the power supply interface. The hardware module determines that the input voltage is greater than a fourth threshold and instructs to start detection, otherwise no detection is performed.

16. The detection device used in the sample detection method for nucleic acid qualitative analysis based on PCR according to any one of claims 12 to 14, characterized in that: The detection device includes a fast charging deception protocol chip and a power supply interface. The power supply voltage is obtained through the fast charging deception protocol chip and the power supply interface. The hardware module determines that the input voltage is greater than a fourth threshold and instructs to start detection, otherwise no detection is performed.

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